Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations

Adam, Virgile; Lelimousin, Mickaël; Boehme, Susan; Desfonds, Guillaume; Nienhaus, Karin; Field, Martin J.; Wiedenmann, Joerg; McSweeney, Seán; Nienhaus, G. Ulrich; Bourgeois, Dominique

doi:10.1073/pnas.0805949105

Cited by 217 publications

(302 citation statements)

References 38 publications

Supporting

Mentioning

277

Contrasting

Unclassified

Order By: Relevance

“…However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued.…”

mentioning

confidence: 99%

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

et al. 2011

View full text Add to dashboard Cite

VOLUME 29 NUMBER 10 OCTOBER 2011 nature biotechnology A r t i c l e sFluorescent proteins (FPs) 1 whose fluorescence can be reversibly or irreversibly switched by optical irradiation have opened new opportunities for the imaging of cells. They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable 11-13 can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths. However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued. RESULTS Generation of the RSFP DreiklangNumerous GFP variants exhibit some degree of (generally undesirable) reversible photoswitching 4,23,24 . We found that the fluorescence of the yellow fluorescent protein Citrine 25,26 , a derivative of GFP, can be reversibly modulated to a small extent by alternate irradiation with light of 365 nm (on switching) and 405 nm (off switching), whereas fluorescence is excited at 515 nm. However, the achievable contrast was low, especially at pH values >6, rendering the reversible switching of Citrine unusable (Supplementary Fig. 1).To further develop this unusual switching behavior, we performed extensive random mutagenesis as well as directed PCR-mediated mutagenesis on a plasmid encoding Citrine. We transformed Escherichia coli with the plasmid, and screened with an automated home-built fluorescence microscope for bacterial colonies expressing fluorescent proteins whose fluorescence was excited with green light (515 nm) and which could be reversibly photoswitched from a fluorescent state to a long-lived nonfluorescent state by irradiation with near-UV (405 nm) light and back to a fluorescent state by UV (365 nm) light (Fig. 1a). In several consecutive screening rounds ~70,000 individual clones were analyzed. Finally, we identified a mutant differing from Citrine at four positions (Citrine-V61L, F64I, Y145H, N146D) ( Supplementary Fig. 2), which can be effectively switched and excited to fluoresce. We named this switchable fluorescent protein Dreiklang, the German word for a three-note chord in music.At thermal equilibrium, Dreiklang adopts the brightly fluorescent ...

show abstract

mentioning

confidence: 99%

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

et al. 2011

View full text Add to dashboard Cite

show abstract

“…They feature the chemically unaltered p-HBI chromophore of GFP. Polar interactions of the chromophore and its surrounding residues affect the charge distributions in the ground and electronically excited states and result in blue shifted emission (29)(30)(31)(32). The yellow emission of zFP538 from Zoanthus sp.…”

Section: Modifications Of the Gfp Chromophore And Its Environment Promentioning

confidence: 99%

“…In a third group of FPs, cistrans isomerisation of the chromophore is responsible for a reversible switching between bright and dark states of the chromophore (32,47,68,69). Usually, the dark chromophore adopts a trans, noncoplanar conformation, whereas the bright state is associated with a cis, planar conformation (32,47). Reversibly switchable chromophores are usually found in engineered green and red FPs (70)(71)(72)(73), but also some natural GFP-like proteins feature switchable chromophores such a cerFP512 from a deep sea cerianthid (74).…”

Section: Light-induced Activation Of the Chromophorementioning

confidence: 99%

“…Thereby, the conjugated p-electron system is extended in the imidazole sidechain of histidine. In a third group of FPs, cistrans isomerisation of the chromophore is responsible for a reversible switching between bright and dark states of the chromophore (32,47,68,69). Usually, the dark chromophore adopts a trans, noncoplanar conformation, whereas the bright state is associated with a cis, planar conformation (32,47).…”

Section: Light-induced Activation Of the Chromophorementioning

confidence: 99%

“…Reversibly switchable chromophores are usually found in engineered green and red FPs (70)(71)(72)(73), but also some natural GFP-like proteins feature switchable chromophores such a cerFP512 from a deep sea cerianthid (74). IrisFP combines two photoactivation processes in one FP: It can be photoconverted from a green to a red fluorescent form by irradiation with 400 nm light but both, the green and the red chromophore can be reversibly switched off by irradiation with blue and green light, respectively (32). An overview of photoconvertible and photoswitchable proteins is given in Table 2.…”

Section: Light-induced Activation Of the Chromophorementioning

confidence: 99%

See 2 more Smart Citations

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

Wiedenmann¹,

Oswald²,

Nienhaus³

2009

IUBMB Life

223

162

View full text Add to dashboard Cite

SummaryThe green fluorescent protein (GFP) from the jellyfish Aequorea victoria can be used as a genetically encoded fluorescence marker due to its autocatalytic formation of the chromophore. In recent years, numerous GFP-like proteins with emission colors ranging from cyan to red were discovered in marine organisms. Their diverse molecular properties enabled novel approaches in live cell imaging but also impose certain limitations on their applicability as markers. In this review, we give an overview of key structural and functional properties of fluorescent proteins that should be considered when selecting a marker protein for a particular application and also discuss challenges that lie ahead in the further optimization of the glowing probes.

show abstract

Super‐Resolution Microscopy: A Comparative Treatment

et al. 2012

View full text Add to dashboard Cite

One of the fundamental limitations of optical microscopy is that of diffraction, or in essence, how small a beam of light can be focused by using an optical lens system. This constraint, or barrier if you will, was theoretically described by Ernst Abbe in 1873 and is roughly equal to half the wavelength of light used to probe the system. Many structures, particularly those within cells, are much smaller than this limit and thus are difficult to visualize. Over the last two decades, a new field of super-resolution imaging has been created and been developed into a broad range of techniques that allow routine imaging beyond the far-field diffraction limit of light. In this unit we outline the basic principles of the various super-resolution imaging modalities, paying particular attention to the technical considerations for biological imaging. Furthermore, we discuss their various applications in the imaging of both fixed and live biological samples.

show abstract

Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations

Cited by 217 publications

References 38 publications

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

Super‐Resolution Microscopy: A Comparative Treatment

Contact Info

Product

Resources

About